Maleic anhydrides are used in medicinal and formulation chemistry for labile covalent linkage between molecules of interest. Introduction of alkyl substituents at positions 2 and 3 of maleic anhydride shifts equilibrium of reaction with aliphatic amines towards free amine and maleic anhydride at pH<7.

At pH>7, the reaction is driven toward the maleamate (maleamic acid when carboxyl group is protonated). At acidic pH, the reaction is driven towards the anhydride and the free amine. This property is useful because in mammals, the pH of blood is about 7.4 (slightly alkaline) while certain intracellular compartments, such as endosomes and lysosome, are acidic (pH<6).
The rate of conversion of maleamates to amines and maleic anhydrides is strongly dependent on substitution at positions 2 and 3 (R1 and R2) of the maleic anhydride system. When R1 and R2 are both hydrogen (maleic anhydride) the reaction is nearly irreversible. Substitution of a single alkyl group at position R1 or R2 (e.g., citraconic anhydride) increases the rate of the reverse reaction 50-fold higher compared to maleic anhydride. At pH 5, the half life for cleavage of a mono-substituted maleamate to yield the free amine and the anhydride is about 8 to 24 h. This half-life is too slow for delivery systems where rapid lability is important. For disubstituted maleamates, steric repulsion of the 2,3 aliphatic groups drives the reaction toward ring closure to form the anhydride and free amine (Kirby Adv. Phys. Org. Chem. 1980, 17, 183-278; Kirby et al. J Chem Soc., Perkin Trans. 2, 1972, 1206-1214). Alkyl substitutions at both R1 and R2 (e.g., 2,3-dimethylmaleic anhydride) increase the rate of the reverse reaction 10,000-fold compared to maleic anhydride. Half-life of cleavage of a disubstituted maleamate from a polyamine is about 5 min at 37° C. and pH 5.5.
The reverse reaction for 2-propionic-3-methylmaleamic acid has been observed to be the same as that for 2,3-dimethylmaleamic acid. We have previously described the use of 2-propionic-3-methylmaleic anhydride (CDM) derivatives for reversible modification of amine-containing polymers (Rozema et al. Proc. Natl. Acad. Sci. USA, 2007, Vol. 104, No. 32, p. 12982-12966). However, the half-life for dialkyl-substituted maleamates can be too energetically favorable, i.e. amide cleavage can occur too rapidly even near neutral pH, for certain in vivo labile delivery systems. At pH 7.5 and 37° C., cleavage of the amide bond in 2,3-dialkyl maleamates to yield anhydrides and free amines occurs with a t1/2 of about 4 h. This rate is too rapid for applications in which longer circulation time is desired. Thus, there is a need for physiologically labile bonds which are more stable in circulation yet retain rapid reversibility in the pH 6 environment of a cell endosome.
It has been shown that connecting alkyl substitutions into a cycle decreases the rate of ring closure reaction in 2,3-dialkyl maleamate. For example, amine release from mono N-methylamido derivative formed from 4,5,6,7-terahydrobenzo[c]furan-1,3-dione and methylamine has kobs=3.5×10−2 (Kirby Adv. Phys. Org. Chem. 1980, 17, 183-278), is 17.5 times faster than for respective 2-methylmaleamate and 32 times slower than for 2,3-dimethylmaleamate. For benzo[c]furan-1,3-dione, introduction of alkyl groups at positions 4 and 7 shifts equilibrium in water toward ring closure. K=k1/k−1 in this equilibrium is 1.5, compare with K=5.3 for the same equilibrium measured for dimethyl maleic acid, or K=102 for phthalic acid itself (Eberson et al. J. Am. Chem. Soc. 1971, Vol. 93, No. 22, p. 5821-5826).

We describe here new disubstituted maleic anhydrides which yield maleamates having half-lives that are shorter than mono-substituted maleamates but slower than previously described dialkyl-substituted maleamates. These new anhydrides, with their slower rate of amide cleavage, achieve longer circulation times in vivo and extended shelf life relative to formulations using previously described disubstituted maleic anhydrides.